The concept of bioorthogonal chemistry is summarized by Carolyn Bertozzi et al. in 2003 (Sletten, Ellen M.; Bertozzi, Carolyn R. “From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions” Acc. Chem. Res., 2011, 44 (9), pp 666-676). It refers to any chemical reaction that can occur inside of living systems without interfering with native biochemical processes. Some bioorthogonal compounds react only with specific unnatural molecules injected from outside, without reacting with biomolecules in the living system. The use of bioorthogonal chemistry typically proceeds in two steps as follows. First, a cellular substrate is modified with a bioorthogonal functional group and introduced into a cell. The cellular substrate may be a metabolite, an enzyme inhibitor, etc. The bioorthogonal functional group must not alter the structure of the substrate dramatically to avoid affecting its bioactivity. Secondly, a probe having a complementary functional group reacting with the bioorthogonal functional group is introduced to react with and label the substrate.
Recently, the bioorthogonal chemistry is paving the way for many novel innovations in the biological field. Direct chemical reactions applicable in living systems with both bioorthogonality and biocompatibility have garnered much attention from both chemists and biologists. For example, although the Staudinger ligation of azides with phosphines represented by Scheme 1 exhibits bioorthogonality under both in vitro and in vivo conditions, its wide application is restricted due to slow reaction kinetics.

Bertozzi et al. have developed many chemicals for biocompatible copper-free click chemistry by focusing on ring-strained alkyne groups as the counterparts to azide groups for increased reactivity. Weissleder et al. have also developed a tetrazine cycloaddition reaction which is extremely fast and highly specific.
In particular, the reason why copper-free click chemistry is required is because, although the classic azide-alkyne cycloaddition using a copper catalyst is a very fast and effective reaction for bioconjugation, it is not suitable for use in live cells due to the toxicity of Cu(I) ions.
As shown in FIG. 5, Bertozzi et al. have developed a bioorthogonal copper-free click chemical reaction requiring no copper catalyst. The reaction is a strain-promoted alkyne-azide cycloaddition.
The potentiality of bioorthogonal chemistry has been proven in many applications. In particular, bioorthogonal chemistry has shown powerful applications in biological fields in combination with metabolic glycoengineering. Through metabolic glycoengineering, unnatural glycans are introduced into cells by feeding specific precursors on the basis of their intrinsic metabolism. Bertozzi's group has pioneered this special technique and demonstrated that modified functional groups can be introduced for bioorthogonal chemistry using the technique. Specifically, the technique has been excellently applied for various purposes, including analysis of cellular glycans, 3D cellular assembly, exploration of metabolic pathways and spatiotemporal imaging of zebrafish development. However, there are few reports on in vivo studies of vertebrates and researches in this field are less active than those in cellular level.
Meanwhile, nanoparticles have emerged as a promising tool in the biomedical field, in which they serve as delivery carriers of imaging agents or nanodrugs. Active targeting is the typical method of improving the specificity of the nanoparticles to disease sites. For active targeting, biological targeting moieties such as antibodies, aptamers or peptides capable of binding to proper receptors on the surface of target cells are used. However, since the number of the receptors binding to the materials is limited, the capacity of the targeting nanoparticles is limited when the receptors are saturated. In addition, because targetable receptors are rarely unique to the disease, the nanoparticles may accumulate in other healthy tissues through these receptors, resulting in reduced therapeutic efficacy or unintended side effects.